The present invention relates to ongoing efforts to achieve controlled drug release and drug targeting to specific tissues, particularly in the area of cancer chemotherapy. More particularly, the invention relates to targeted drug delivery by means of intact bacterial minicells, which are able to deliver drugs intracellularly, within desired target cells in-vivo and in-vitro. Minicells containing chemical or biochemical drugs constitute novel delivery vehicles, capable of being targeted to specific cells. One method of targeting these vehicles employs bispecific molecules that specifically bind to both a minicell surface structure and a target cell surface structure, such as a receptor. The bispecific ligands mediate an interaction between the minicells and target cells, such that the target cells engulf the minicells, which release their drug payload into the cytoplasm of the target cells. Once cytoplasmically released, the drug acts on intracellular targets, such as intracellular organelles, the nucleus, the cytoskeleton, enzymes, and co-factors, to achieve a therapeutic effect. In another method of drug delivery, phagocytosis- or endocytosis-competent target cells engulf drug-loaded minicells without the use of bispecific ligands.
Currently, most drugs used for treating cancer are administered systemically. Although systemic delivery of cytotoxic anticancer drugs plays a crucial role in cancer therapeutics, it also engenders serious problems. For instance, systemic exposure of normal tissues/organs to the administered drug can cause severe toxicity (Sarosy and Reed, 1993). This is exacerbated by the fact that systemically delivered cancer chemotherapy drugs often must be delivered at very high dosages to overcome poor bioavailability of the drugs and the large volume of distribution within a patient. Also, systemic drug administration can be invasive, as it often requires the use of a secured catheter in a major blood vessel. Because systemic drug administration often requires the use of veins, either peripheral or central, it can cause local complications such as phlebitis. Extravasation of a drug also can lead to vesicant/tissue damage at the local site of administration, such as is commonly seen upon administration of vinca alkaloids and anthracyclines.
Because existing systems for targeted drug delivery are seriously deficient, current cancer drug treatment strategies poorly address the problems that attend systemic drug administration. One approach for addressing these problems involves simply modifying administration schedules or infusion regimens, which may be either bolus, intermittent, or continuous. This approach, however, provides very limited benefits.
Some alternative approaches to intravenous injection also exist, each designed to provide regional delivery, i.e., selective delivery to a tumor region. Examples of such alternatives include polymeric implants, intra-peritoneal infusion, intra-pleural infusion, intra-arterial delivery, chemo-embolization, and inhalation of aerosols. In particular, intra-peritoneal administration of chemotherapy has been studied extensively for ovarian carcinoma and other abdominal tumors (Kirmani et al., 1994; Alberts et al., 1996). Unfortunately, each of these delivery methods, including intra-peritoneal administration, has achieved only marginal success at selectively delivering drugs to a tumor site and reducing side effects.
Other attempts to address the problems with systemic delivery of cytotoxic anticancer drugs include the use of alternative drug formulations and delivery systems, including controlled-release biodegradable polymers, polymeric microsphere carriers and liposomes, as well as the co-administration of cytoprotective agents with antineoplastics. Chonn and Cullis, 1995; Kemp et al., 1996; Kumanohoso et al., 1997; Schiller et al., 1996; Sharma et al., 1996; Sipos et al., 1997.
The use of liposomes as drug carriers for chemotherapeutic agents originally was proposed as a means for manipulating drug distribution to improve anti-tumor efficacy and to reduce toxicity (reviewed by Allen, 1997). Through encapsulation of drugs in a macromolecular carrier, such as a liposome, the volume of distribution is significantly reduced and the concentration of drug in a tumor is increased. This causes a decrease in the amounts and types of nonspecific toxicities, and an increase in the amount of drug that can be effectively delivered to a tumor (Papahadjopoulos and Gabizon, 1995; Gabizon and Martin, 1997; Martin, 1998). Liposomes protect drugs from metabolism and inactivation in plasma. Further, due to size limitations in the transport of large molecules or carriers across healthy endothelia, drugs accumulate to a reduced extent in healthy tissues (Mayer et al., 1989; Working et al., 1994).
To prolong their circulation time, liposomes are coated with polyethylene glycol (PEG), a synthetic hydrophilic polymer (Woodle and Lasic, 1992). The PEG headgroup serves as a barrier, sterically inhibiting hydrophobic and electrostatic interactions with a variety of blood components and plasma opsonins at the liposome surface, and thereby retards recognition of liposomes by the reticuloendothelial system. PEG-coated liposomes are termed “sterically stabilized” (SSL) or STEALTH liposomes (Lasic and Martin, 1995). This technology gave rise to a commercial pharmaceutical formulation of pegylated liposomal Doxorubicin, known as Doxil in the U.S. and Caelyx in Europe. A wide array of other drugs also have been encapsulated in liposomes for cancer treatment (Heath et al., 1983; Papahadjopoulos et al., 1991; Allen et al., 1992; Vaage et al., 1993b; Burke and Gao, 1994; Sharma et al., 1995; Jones et al., 1997; Working, 1998).
Liposomal drug carriers, unfortunately, have several drawbacks. For example, in vivo, drugs often leak out of liposomes at a sufficient rate to become bioavailable, causing toxicity to normal tissues. Similarly, liposomes are unstable in vivo, where their breakdown releases drug and causes toxicity to normal tissues. Also, liposomal formulations of highly hydrophilic drugs can have prohibitively low bioavailability at a tumor site, because hydrophilic drugs have extremely low membrane permeability. This limits drug release once liposomal carriers reach a tumor. Highly hydrophobic drugs also tend to associate mainly with the bilayer compartment of liposomes, causing low entrapment stability due to rapid redistribution of a drug to plasma components. Additionally, some drugs, such as 1-β-D-arabinofuranosylcytosine (ara-C) and methotrexate, only enter tumor cells directly, via membrane transporters Plageman et al., 1978; Wiley et al., 1982; Westerhof et al., 1991, 1995; Antony, 1992). In such cases, a liposomal carrier would need to release sufficient drug near a tumor site to achieve a therapeutic effect (Heath et al., 1983; Matthay et al., 1989; Allen et al., 1992). Lastly, the use of conventional liposome formulations increases a patient's risk of acquiring opportunistic infections (White, 1997), owing to localization of drugs in reticuloendothelial system macrophages and an attendant macrophage toxicity (Allen et al., 1984; Daemen et al., 1995, 1997). This problem becomes accentuated in immune deficient patients, such as AIDS patients being treated for Kaposi's sarcoma.
Because problems continue to hamper significantly the success of cancer therapeutics, an urgent need exists for targeted drug delivery strategies that will either selectively deliver drugs to tumor cells and target organs, or protect normal tissues from administered antineoplastic agents. Such strategies should improve the efficacy of drug treatment by increasing the therapeutic indexes of anticancer agents, while minimizing the risks of drug-related toxicity.
An international patent application, PCT/IB02/04632, has described recombinant, intact minicells that contain therapeutic nucleic acid molecules. Such minicells are effective vectors for delivering oligonucleotides and polynucleotides to host cells in vitro and in vivo. Data presented in PCT/IB02/04632 demonstrated, for example, that recombinant minicells carrying mammalian gene expression plasmids can be delivered to phagocytic cells and to non-phagocytic cells. The application also described the genetic transformation of minicell-producing parent bacterial strains with heterologous nucleic acids carried on episomally-replicating plasmid DNAs. Upon separation of parent bacteria and minicells, some of the episomal DNA segregated into the minicells. The resulting recombinant minicells were readily engulfed by mammalian phagocytic cells and became degraded within intracellular phagolysosomes. Surprisingly, some of the recombinant DNA escaped the phagolysosomal membrane and was transported to the mammalian cell nucleus, where the recombinant genes were expressed. Thus, the application showed a usefulness for minicells in human and animal gene therapy.
The present invention builds on these recent discoveries relating to minicells, and addresses the continuing needs for improved drug delivery strategies, especially in the context of cancer chemotherapy.